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Effects of Bt maize containing three Bt proteins on earthworms

(2008 – 2011) RWTH Aachen University, Institute of Biology III (Plant Physiology), Worringer Weg 1, 52074 Aachen

Topic

The aim of this project is to investigate the potential effects of the genetically modified Bt maize cultivar MON89034xMON88017 on earthworms. This maize produces three different Bt proteins, making it resistant to both the European corn borer and the Western corn rootworm.

Earthworms are a particularly important group of organisms in the soil ecosystem. When genetically modified crops are grown, earthworms could come into contact with Bt proteins either by absorbing plant material in the soil or via root exudates. Up until now, very little research has been undertaken to examine the effect of these proteins on earthworms.

This project will investigate the following questions:

  • Is the incidence and density of earthworms in trial plots of genetically modified maize lower than in plots of conventional maize?
  • Is there a difference between the conventional varieties in this respect and if so, how great is it?
  • How much Bt protein is ingested by earthworms in GM maize fields?
  • Does the absorption of Bt proteins impair the development and vitality of earthworms?

Experiment description

The genetically modified variety, its isogenic parent variety and two further conventional control varieties of maize were grown in a field trial. This method enables researchers to distinguish between potential Bt effects and varietal effects. In addition, on some of the plots, the isogenic variety was treated with an insecticide so that the effect of conventional control methods could also be included in the study.

Before sowing and after harvest, soil was removed from an area of 0.45 x 0.45 cm on each plot down to a depth of 20 cm …

… and examined for earthworms.

Captured earthworms were counted and identified.

An earthworm ‘escape test’ was carried out in the laboratory: Untreated soil was placed in one half of the pot, while the other half was filled with soil containing Bt protein. Ten earthworms were placed on the dividing line of each pot.

Determining earthworm diversity and density

Following a standardised procedure, earthworms were driven out and counted in spring before sowing the maize and again in autumn following the maize harvest. Collected earthworms were measured, identified and classified according to three ecological habitats – surface litter, upper mineral soil and deeper soil layers.

Determining the exposure of earthworms to Bt proteins

The ELISA detection method was used to measure the amount of Bt protein detectable in the captured earthworms. This enables researchers to estimate the extent to which these organisms are exposed to it.

Laboratory trials to determine the potential effects of Bt proteins on earthworms

Long-term trial with earthworms: In long-term trials, earthworms were kept in controlled conditions (greenhouse or phytochamber) in pots of Bt maize or were fed Bt plant material. The growth, weight and mortality rate of the worms were studied. The aim here was to cover the entire lifespan of earthworms over very extensive trial periods.

Earthworm escape test: Earthworms were placed in big containers – one half containing untreated soil and the other filled with soil containing Bt protein. The Bt proteins were added individually and in combination. The number of worms in each half was counted after 72 hours.

Results

Determining earthworm diversity and density

Initial earthworm samples were taken from selected plots and various areas of the buffer planting zone in spring 2008 before sowing, in order to obtain a baseline for the existing earthworm population before the start of the trial. A total of 350 worms (266 intact worms and 84 worm fragments) were found, representing an average of 13.46 worms (10.23 intact worms and 3.23 worm fragments) per sample area.

Earthworm samples were taken before sowing and after harvest each season. Overall, the distribution of earthworms on the field was very heterogeneous and the density was low. For this reason, the sampling dates from autumn 2008 to autumn 2010 were added together and subjected to a statistical analysis.

In summary, the Bt maize variety was not found to have any impact on earthworms.

Determining the exposure of earthworms to Bt proteins

The ELISA detection method was used to check whether the various Bt proteins are present in earthworms. Two of the three Bt proteins were not detected.

Laboratory trials to determine the potential effects of Bt proteins on earthworms

Two repetitions of the long-term earthworm trial were conducted. In both repetitions, no more than a quarter of the earthworms used were recaptured. Since this low recapture rate was the same for all varieties, no further statistical analysis was carried out.

The earthworm escape test was conducted using a representative species. No avoidance response was detected. The earthworms were evenly distributed between the two halves of the containers, both when the Bt proteins were added individually, and when combinations of several Bt proteins were added. The Bt protein concentrations were between 100 and 1000 times higher than the Bt protein levels found in the trial field. Mixing in fresh or dried maize leaves did not have any effect.